Surgical resection, chemotherapy, radiotherapy and immunotherapy have been mainly used alone or together as a treating method of cancer (malignant tumor). Although the immunotherapy is still in a developmental stage, it has a lot of potentialities and is therefore expected to make further progress in the near future.
The immunotherapy specific to the cancer means a treating method in which a cytotoxic activity is affected only upon cancer cells. As a drug showing the cytotoxic activity is combined with an antibody to have directivity in this therapy, it is now called a “missile therapy.” Studies have now been carried out to find an antigen that will be effectively used for the preparation of the antibody with a minimum of side effects by targeting as the antigen a substance that is abnormally expressed in the cancer cells or that will change according to malignant alternation or canceration of cells. Such antigen is called a cancer-associated antigen.
Among antibodies with multiple specificity, an antibody with bispecificity (Bispecific Antibody: BsAb) is now studied intensively. The bispecific antibody can bind specifically to two different kinds of antigens so that it will be utilized as a therapeutic agent having a specific anti-cancer effect. A diabody is a minimum unit of the above bispecific antibody. It was developed by utilizing the property that the variable region in the heavy chain (VH) and the variable region in the light chain (VL) derived from the same parent antibody will form a hetero-dimer through non-covalent bond (Hollinger, et al., Proc. Natl. Acad. Sci. USA 90, 6444-6448, 1993).
A diabody-type bispecific antibody is characterized by having low immunogenicity and high infiltrating activity into tumor tissues due to its low molecular weight (ca. 60,000), and by being easily mass-produced at a low cost with use of microorganisms such as E. coli, and by being easily altered in function by means of genetic engineering.
It is therefore strongly desired to develop the diabody-type bispecific antibody that may resolve problems encountered in the conventional cancer therapies, especially the problems that it is impossible to completely remove cancer cells located at a site where the structure of tissues is complicated and that there is no way to remove the remaining cancer cells after a surgical operation.
Up to now, as an example of the diabody-type bispecific antibody, a fused protein has been developed. It has bispecificity derived from an antibody against a glycoprotein MUC1 often observed on adenocytes and an anti-CD3 antibody, and further contains a super antigen such as cellular enterotoxin in the same polypeptide as the variable region of the antibody (PCT Publication No. WO02/06486).
However, since the conventional diabody-type bispecific antibodies are a recombinant protein produced with use of microorganism, they tend to be inferior in effectiveness and stability after administration to the bispecific antibodies chemically synthesized. The conventional diabody-type bispecific antibodies also have some problems or disadvantages such as that they have to be produced in a high concentration in order to obtain the same effects as those by the bispecific antibodies chemically synthesized.
On the other hand, it was already reported that an antibody specific to HER-2/neu(c-erb-2), one of epidermal growth factor receptors (EGFR) that are a cell membrane-bound glycoprotein, could reduce the tumor by about 20% in breast cancer patients.
The present inventors have studied to resolve the above problems so as to develop a novel diabody-type bispecific antibody that shows an excellent effect, and finally succeeded in preparing a diabody-type bispecific antibody showing a more remarkable effect than the conventional diabody-type bispecific antibodies and chemically synthesized bispecific antibodies even in a very low concentration and in the absence of the super antigen.